Detection of α-syn pores.

<p><b>A</b>) Pore formation results in increased current-flows over the membrane (black trace) compared to intact bilayers (grey trace) when a voltage ramp is applied. <b>B</b>) Pore detection rate for oligomers obtained with 2.1 µM α-syn with 1% DMSO and 20 µM Fe³⁺ at RT. 4 and 24 h of incubation did not lead to any pore detections (N = 8, each). In 33% of all 48 h-samples (N = 9) pore formations could be detected, increasing to a maximum at 72 h (N = 10). Further incubation results in a decrease of pore detection (N = 10). Non-incubated α-syn monomers did not lead to pore detections (N = 4), as well as DMSO and Fe³⁺ (“buffer”) after all tested incubation times (N = 4, each). α-hemolysin was used as a positive control (α-HL; N = 4). <b>C</b>) In a further set of experiments, the effects of different α-syn concentrations and the effect of the aggregation inhibitor baicalein were investigated after incubation of α-syn for 72 h with DMSO and Fe³⁺. Shown is the probability of pore detection following sequential application of aliquots of the respective samples to the bilayer. α-syn was used at 7.0 µM (N = 69), 2.1 µM (N = 35) and 0.7 µM (N = 8) with up to 9 aliquots applied per sample. With decreasing α-syn concentration, cumulative pore detection rate decreases significantly (p<0.001). Co-incubation of 2.1 µM of α-syn with 50 µM of baicalein (N = 8) significantly decreases pore detection compared to 2.1 µM control condition (p<0.005). <b>D</b>) When different voltages were clamped to membranes with inserted pores, step-like changes in conductivity were consistently observed during the duration of the voltage-pulse.</p

Schematic illustration of different models for increased membrane permeability caused by α-syn oligomers.

<p>In principle, α-syn oligomers could cause increased conductance of lipid membranes by different modes of action. <b>A</b>) A diffuse damage of the bilayer could lead to an unspecific increase in transmembrane current flow. <b>B</b>) Distinct pores could be formed in the bilayer that switch between two or more different conformational states, resulting in corresponding changes in conductivity. <b>C</b>) Different numbers of uniform pores could spontaneously insert and de-insert into the membrane leading to step-like changes in conductivity. <b>D</b>) The number of “open” pores could fluctuate due to open and closure events of permanently inserted pore complexes.</p

Influence of metal-ion induced asyn oligomer formation and pseudophosphorylation on membrane binding.

<p>A. FIDA analysis (upper panel) and 2D FIDA histograms (lower panel) demonstrate that Fe³⁺/Al³⁺ induced asyn⁶⁴⁷ oligomers show a high affinity to POPC-SUV⁴⁸⁸. Conversely, membrane binding of asyn129E⁶⁴⁷ oligomers is nearly abolished for Fe³⁺ and markedly reduced for Al³⁺ induced oligomers. B. SIFT segment analysis also demonstrates an increased mixed particle signal for asyn⁶⁴⁷ as compared to asyn129E⁶⁴⁷ (“bound oligomer”, corresponding to SIFT segments 4–15; also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098906#pone-0098906-g001" target="_blank">figure 1A</a>.), while levels of free oligomers are higher in asyn129E⁶⁴⁷ (“oligo”, corresponding to segments 16–18). Levels of significance are depicted as * = p<0.05, ** = p<0.01; n = 3.</p

Schematic illustrating the influence of mimicking Ser129 phosphorylation on asyn membrane interactions.

<p>A. For stressed gel-state membranes (DPPC-SUV), mimicking phosphorylation at Ser129 mildly reduces asyn binding affinity. B. Both pseudophosphorylated and unphosphorylated asyn monomers show no affinity to membranes in the liquid-crystalline state (POPC). C. Fe³⁺ induced asyn oligomers show a high membrane affinity and potentially act as membrane pores. Pseudophosphorylation at Ser129 shows a differential influence on asyn aggregation and binding behaviour. While increasing oligomer formation in presence of trivalent metal-ions, Ser129 pseudophosphorylation inhibits membrane binding and may thus allow oligomer sequestration into larger aggregates such as fibrils.</p

Effect of Ser129 pseudophosphorylation on asyn monomer binding to lipid vesicles.

<p>aSyn⁶⁴⁷ and asyn129E⁶⁴⁷ were coincubated with POPC- and DPPC-SUV⁴⁸⁸. Schematic A. demonstrates the appearance of vesicles, protein monomers and oligomers in 2D FIDA histograms (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098906#pone.0098906-Hogen1" target="_blank">[7]</a>). B. aSyn⁶⁴⁷ and asyn129E⁶⁴⁷ monomers show no interactions with POPC-SUV. Conversely, extensive monomer binding to DPPC-SUV is seen irrespective of pseudophosphorylation status. C. Quantitative FIDA analysis shows a mild tendency towards reduced vesicle binding of asyn129E⁶⁴⁷. D. Quantitative 2D-FIDA analysis of monomers bound to DPPC-SUV demonstrates an overall higher amount of bound monomers (total brightness), determined through brightness and concentration of bicolored particles. Furthermore, the higher fluorescence intensity of the bicolored particles (single vesicle brightness) indicates that more monomers are bound per vesicle. E. In a control experiment, asyn⁶⁴⁷ and asyn129E⁶⁴⁷ particle brightness (Q) as determined by 1D FIDA analysis (one component fit) is unchanged in presence of POPC-SUV as compared to control measurements in the absence of vesicles, confirming that no membrane binding takes place. However, Q increases in presence of DPPC-SUV. Dissolution of DPPC-SUV by SDS yields an asyn⁶⁴⁷ particle brightness similar to monomeric asyn⁶⁴⁷, indicating that oligomer formation is not induced by binding to DPPC-SUV. Levels of significance are depicted as * = p<0.05; n = 3.</p